**3.1 Harm to human**

#### **3.1.1 Carcinogenecity**

Aflatoxins are the most notorious within mycotoxins. These toxins target liver of human and animal and can lead to hepatic cancer and even death. Among them, the AFB1 was categorized as No. 1 carcinogen by International Agency for Research on Cancer (IARC) in 1988. A significant negative relationship between the amount of aflatoxin in food with incidence of liver cancer was observed (Gao, 1998).

#### **3.1.1.1 Hazards**

To explore the epidemiological feature, as well as the changing rule of the morbidities of malignant diseases, especially, liver cancer, in the population of Fushui county, Guangxi province in the period of 1997~2003 (Huang & Wei, 2006), morbidity data of all malignant diseases in Fushui county, Guangxi province, were collected. Population data were collected as well. The population construction, by ages and sexes, was calculated, referring to the data of overall survey in 1990. They calculated statistically the yearly rates of liver cancer, in order to produce the changing trends comparing the data with history data. Results showed the mean morbidity rate of liver cancer in Fushui county was 52.79/105 (or 50.50/105, if adjusted to the Chinese population, 1964). Liver cancer morbidity rate was the highest in all of the malignant diseases occupying 57.01% of the morbidity rate of overall malignant diseases. Male is more susceptible than female. The ratio of morbidity rate between men and women was 4.93:1. Morbidity rates of liver cancer rose by ages, with the mid-age of 47.58. Morbidity rates of liver cancer in these years remained relatively stable. Comparing with 1970's data, these rates seemed already slightly reduced. Considering that mortality rates (a replacement of cancer morbidity rates) of liver cancer already rise obviously in Guangxi, as well as in the rest area of China. The trend that morbidity of liver cancer in Fusui county reduces a little comparing with historical data. This could be considered as a reflection of the effect of cancer control, which had already been being carried out in Fusui county. The facts that mid-age group of morbidity is decreased and that morbidities in younger age group is significantly reduced are the important evidences of the effect of the field cancer control.

In 2008,polymorphism studies on CYP3A5 genes in 210 patients from high aflatoxin contamination area showed that about 60% of the total individuals are those with high level CYP3A5 expression[25]. This percentage is far higher than that has been reported in Guangdong, Hunan provinces and Singapore, which are considered low aflatoxin B1 contamination. Consequently, the contamination of aflatoxin is the main reason of the occurrence of liver cancer in this area (Lu *et al.*, 2008). In the same year, studies on the relationships of the aflatoxin exposure, glutathione transferase gene polymorphism and high risk group with primary hepatocellular carcinoma shown that the exposure of aflartoxin is the main risk factor of the occurrence of liver cancer in this area (Tang *et al.*, 2008).

In 1970~1999, there were 4,215 new liver cancer cases in Zhongshan. Its crude incidence rate, China and world standardized rates were 13.0/105, 12.5/105, 16.8/105,respectively. There is no increasing or decreasing trend for its incidence rates in 1975~1994. However, a declining tendency between 1995 and 1999 was observed. The liver cancer incidence rate during this period in Zhongshan was moderate comparing with the worldwide statistics, but at middle-high level and at low level compared with urban and rural pilot areas in China at the same time period.

The crude and standardized incidences of liver cancer were analyzed by collecting the disease information from the rural area in Ningbo from 2006 to 2008 (Cui *et al.*, 2009). The results show that the crude incidence of hepatoma of the rural residents in Ningbo from 2006 to 2008 is 38.66/105. The age standardized incidence of this disease is 32.14/105. The incidence of hepatoma increased with age. Its incidence in male is 2.77 times of that in female. As to the diagnosis technology, imageology is the most persuasive method to make a definite diagnosis with a ratio of 58.93%. Next effective method is the pathological examination with a ratio of 36.72%. Hepatoma incidence of rural residents in Ningbo is above the average ratio of that in Zhejiang province and China.

#### **3.1.1.2 Pathogenesis**

24 Aflatoxins – Detection, Measurement and Control

provinces are the high incidence regions, which is significantly higher than the already reported AFB1 contamination area, Guangdong, Hunan and Singapore. It is widely accepted that aflatoxin contamination in the region is correlated well with the onset of liver cancer in human. Studies showed that AFB1 causes the p53 gene mutation in human cancer cell. P53 is a tumor suppressor, a transcription factor involved in the regulation of

Aflatoxins are the most notorious within mycotoxins. These toxins target liver of human and animal and can lead to hepatic cancer and even death. Among them, the AFB1 was categorized as No. 1 carcinogen by International Agency for Research on Cancer (IARC) in 1988. A significant negative relationship between the amount of aflatoxin in food with

To explore the epidemiological feature, as well as the changing rule of the morbidities of malignant diseases, especially, liver cancer, in the population of Fushui county, Guangxi province in the period of 1997~2003 (Huang & Wei, 2006), morbidity data of all malignant diseases in Fushui county, Guangxi province, were collected. Population data were collected as well. The population construction, by ages and sexes, was calculated, referring to the data of overall survey in 1990. They calculated statistically the yearly rates of liver cancer, in order to produce the changing trends comparing the data with history data. Results showed the mean morbidity rate of liver cancer in Fushui county was 52.79/105 (or 50.50/105, if adjusted to the Chinese population, 1964). Liver cancer morbidity rate was the highest in all of the malignant diseases occupying 57.01% of the morbidity rate of overall malignant diseases. Male is more susceptible than female. The ratio of morbidity rate between men and women was 4.93:1. Morbidity rates of liver cancer rose by ages, with the mid-age of 47.58. Morbidity rates of liver cancer in these years remained relatively stable. Comparing with 1970's data, these rates seemed already slightly reduced. Considering that mortality rates (a replacement of cancer morbidity rates) of liver cancer already rise obviously in Guangxi, as well as in the rest area of China. The trend that morbidity of liver cancer in Fusui county reduces a little comparing with historical data. This could be considered as a reflection of the effect of cancer control, which had already been being carried out in Fusui county. The facts that mid-age group of morbidity is decreased and that morbidities in younger age group is significantly reduced are the important evidences of the effect of the field cancer

In 2008,polymorphism studies on CYP3A5 genes in 210 patients from high aflatoxin contamination area showed that about 60% of the total individuals are those with high level CYP3A5 expression[25]. This percentage is far higher than that has been reported in Guangdong, Hunan provinces and Singapore, which are considered low aflatoxin B1 contamination. Consequently, the contamination of aflatoxin is the main reason of the occurrence of liver cancer in this area (Lu *et al.*, 2008). In the same year, studies on the relationships of the aflatoxin exposure, glutathione transferase gene polymorphism and high risk group with primary hepatocellular carcinoma shown that the exposure of aflartoxin is the

main risk factor of the occurrence of liver cancer in this area (Tang *et al.*, 2008).

the cell cycle.

**3.1.1.1 Hazards** 

control.

**3.1 Harm to human 3.1.1 Carcinogenecity** 

incidence of liver cancer was observed (Gao, 1998).

The aflactoxin can result in cancer by a variety of molecular mechanisms.

Aflatoxin exposures can begin in utero and continue through childhood. A mutation in the P53 tumor suppressor gene from AGG to AGT (arginine to serine) transversion at codon 249 (Ser249 mutation) has been reported for hepatocellular carcinoma and matched plasma DNA found in plasma of young children from a region of high aflatoxin exposure (Xu, 2009). This gene mutation in tumor-derived DNA has recently been detected in plasma or serum DNA from adult hepatocellular carcinoma patients. The presence of this mutation before hepatocellular carcinoma onset (e.g., in patients with cirrhosis and patients without clinically diagnosed liver disease) may indicate that the mutation is a marker of chronic exposure to aflatoxin (Kirk *et al.*, 2005). This mutation has been detected in areas with high aflatoxin exposure while it is rare in the low aflatoxin exposure regions (Duan *et al.*, 2005).

A close relationship between the expression of survivin, a newly founded inhibitor of apoptosis protein (IAP), and the abnormality of Wnt signal transduction pathway, was revealed (Ban & Cao, 2005), (Jiao *et al.*, 2007). The HBV is prevalent in high hepatic cancer risk areas, so there is a synergetic effect between the two risk factors. Using populationbased case-control study to find the main risk factors of hepatocellular carcinoma (HCC), when people exposed with three main environmental factors (HBsAg, intake of moldy food and drinking raw water), the ORs of hepatic cancer were increased by several times suggesting a conjugated effect between HbsAg and AFB1 albumin adduct.

Concerning the coordinate cancergenic mechanism between AFB1 and HBV, it is concluded that: i. both of the risk factors can reduce the gene expression level of drug-metabolizing enzyme; ii. Chronic inflammatory reaction increased possibility of p53 mutation induced by AFTB1. iii. Chronic infection of HBV changes AFTB1 to an active form. iv. HBX inhibits the nucleus excision repair of DNA, hindering the repair of AFB1-DNA adduct and similar DNA damage and accelerating the process of carcinomatous change of hepatic cells. Besides, the sensibility of host to AFB1 and fatty degeneration of liver could also come to be carcinogens (Xu, 2009).

Aflatoxin Contamination and Research in China 27

AFG1; the synthesis of AFB2 and AFG2 is: The front part of the process is the same to AFGB1 and AFG1, the difference is versicolorin B changed to dihydro versicolorin, then to dihydro-O-

The factors closely related to the synthesis of aflatoxins include the genes, enzymes and the environmental conditions. The genes related to the aflatoxin biosythesis were analyzed by the technology of gene chip as well as RT-PCR method (Hu & Xu, 2009). Six abnormally expressed genes were detected. The six genes are *aflA*, *aflE*, *aflF*, *aflR*, *aflT* and *aflX*. According to the result, the different expression level of *aflR* has close correlation with the

Some related factors in the synthesis of aflatoxins were studied (Lu *et al.*, 2010). The results show that several dehydrogenases, peroxidases, cyclases, methyltransferases and oxidoreductase have a key role in the biosynthesis of aflatoxins. The activity of those enzymes affected the yield of aflatoxins directly. On the other hand, the most important environmental factors are carbon and nitrogen source, power of hydrogen, temperature,

Studies on resistance to aflatoxigenic fungi through molecular biology in China include: the synthesis of artificial antigens, the aflatoxin resistant microorganism and catabolic enzymes, screening of important resistance genes and molecular markers. Aflatoxins are small molecules, thus the immunization of aflatoxins was achieved through coupling with large proteins. With the m-chloroperbenzoic acid (MCPBA) as oxygenant turning the aflatoxin G1 to 8.9- epoxide, a compound AFG1-BSA was obtained after the epoxide coupling with BSA in a two-phase reaction system (Zhang & Li, 2008). Ultraviolet scanning of the compound showed a significant difference comparing with the scanning result of aflatoxin G1 and a different fluorescence intensity between them, which indicated the coupling of BSA and AFG1. This analytical method promoted the study on the preparation of monoclonal

The mixture of broad bean and wheat flour during fermentation was used to screen antagonistic bacteria against aflatoxigenic *A. flavus* (Gao & Ding, 2010). A strain L4 with strong antifungal activity against the aflatoxin-producing fungus *A. flavus* was selected using agar medium (BAM). According to its morphological, physiological and biochemical characteristics and 16S rRNA gene sequence homology analysis, L4 was identified as *Bacillus subtilis*. When L4 and *A. flavus* were co-cultured for 15 days, the weight of the mycelium and the production of aflatoxin B1 were both significantly lower than those of *A. flavus* cultured without L4. The accumulation of AFB1 was greatly inhibited, the suppression effective ratio was 93.7%. When L4 culture supernatant was mixed with the spore suspension of *A. flavus* at ratio of 1:1 and then inoculated on corn, the germination and

Using the method of filter paper diffusion. a strain of marine microorganism which exhibited highly inhibitory effect on *Aspergillus flavus* was screened (Kong & Liu, 2010). With the aid of 16S rDNA gene sequence, this marine strain was finally identified as a marine strain of *Bacillus megaterium*. Then, its inhibitory effects on mycelium extending, spore germination and aflatoxin biosynthesis of *A. flavus* were further studied. Quantitative analysis kit for aflatoxins (Beacon) was used to determine the concentration of aflatoxin. The

methylsterigmatocystin, to AFB2 and AFG2.

**4.1.2 Factors involved in the synthesis** 

water activity and plant metabolites.

antibody and immunoaffinity column.

growth of *A. flavus* was completely inhibited.

**4.2 The resistant research** 

production of aflatoxin.

#### **3.1.2 Chronic intoxication**

#### **3.1.2.1 Correlated event**

It is widely reported that the aflatoxins cause many human acute intoxication events. For example, farmers in three families ate mildewed rice (the aflatoxin content reached 225.9 μg/kg) in Taiwan province. This event led to 25 persons poisoned and the deaths of three children, among 39 persons involved. There was an explosion of Toxic Hepatitis caused by aflatoxin in 200 villages, 397 persons got the disease and 106 persons dead (Wu, 2007).

#### **3.1.2.2 Symptoms**

After a person ate aflatoxin contaminated food, it may cause fever, abdominal pain, vomiting, more seriously splenohepatomegalia, hepatalgia, skin mucous membrane stained yellow, ascites, edema of lower limbs and dysfunction of liver after 2~3 weeks. The cardiac dilatation, pulmonary edema, coma, spasm may also occur (Xiao & Xing, 2003).

### **3.2 Harm to animals**

The aflatoxin can cause damage to the liver, and the sensitivity of aflatoxin is closely related to animal size, species, gender, age, and nutrition. The aflatoxin can damage to the animal embryo, decrease the liver function, cause a decline in milk and egg production, and decrease animal immunity if infection of micro organism happens repeatedly. During the growth period, animals at young stage are more likely to be infected. The clinical manifestations poisoning include low reproductive capacity, gastrointestinal dysfunction, decline in feed utilization. Moreover, dairy cattle could produce AFM1 and M2.

#### **3.3 Financial Ioss**

According to statistics, the aflatoxins contamination of animal feed in USA led to about 10% financial loss. Besides, the death of livestock results in a severe loss to the agriculture (Zhang, 2008). At the same time, aflatoxins can reduce the production of the food and fiber crops.
